CN109423486A - Novel UDP- glycosyl transferase and its application - Google Patents

Abstract

The present invention relates to novel UDP- glycosyl transferase and its application, the glycosyl transferase can specifically and efficiently be catalyzed the specific site of tetracyclic diterpene compound plus glycosyl, to generate a kind of glycosylated tetracyclic diterpene compound.

Description

Novel UDP- glycosyl transferase and its application

Technical field

The invention belongs to botany and chemical field, more particularly it relates to novel UDP- glycosyl transferase and its Using.

Background technique

Glycosylation modified modification reaction after being the one kind being widely present in natural products, is frequently experienced in biosynthesis pathway Terminal, therefore the physicochemical property and physiological activity of natural products are had an important influence.The activity of many plant origins is secondary Metabolite is present in nature in the form of glucosides.The glycosylation modified reaction of Secondary Metabolite Production in Plants is mainly by uridine Diphosphonic acid (UDP)-glycosyl transferase catalysis occurs.UDP- glycosyl transferase (UGT) is by glycosyl from the glycosyl donor molecule of activation It is transferred on glycosyl acceptor molecule.

Steviol glycoside (Steviol glycosides) is a kind of dammara ene-type tetracyclic diterpene class glycoside compounds, is originated in From compositae plant STEVIA REBAUDIANA (Stevia rebaudiana), have sugariness is high, heat is low, non-toxic, high temperature resistant, acid and alkali-resistance and The advantages that good water solubility, therefore become the outstanding person of natural sweetener, it is raw to be widely used in food, beverage, flavoring agent processing etc. It, can obesity, hypertension, diabetes and saprodontia long-term to avoid traditional sugar material such as sucrose or excessively eating initiation during production is industrial Deng the disease of harm health.European Union can be used as food additives in approval stevioside in 2011 and use.

Steviol glycoside is only reported in four plant species so far, is the STEVIA REBAUDIANA of composite family Stevia respectively S.rebaudiana and Stevia phlebophylla A.Gray, rose family rubus sweetness agent classification Rubus Suavissimus S.Lee and Umbelliferae angelica Angelica Keiskei Angelica keiskei (Miq.) Koidz..Sweetleaf stirrup Son also known as Sweet tea, sweetleaf raspberry etc., originate from the mountain area of China Guangxi Zhuang Autonomous Region, are a kind of rare natural plants.? Southwest China ethnic group is civil long applicating history, have it is clearing heat and detoxicating, promote the production of body fluid, moistening lung, cough-relieving, relieving sore-throat the effect of. Current isolated a variety of stevioside glycosides compounds from sweetness agent classification mainly include sweetness agent classification glycosides (rubusoside accounts for 5% or more leaf dry weight) and suaviosdie A~L, however, this field is not obtained from the species still Valuable glycosyl transferase does not know about the accurate synthesis mechanism of each stevioside glycosides compound in the species yet.

With the progress of last decade synthetic biology technology, certain compounds are produced using the heterologous synthetic method of microorganism It is possibly realized, it has the advantages that at low cost, occupied area is few, and product quality is easily-controllable.Therefore, it is necessary to find and reflect for this field Fixed new glycosyl transferase is efficiently produced useful in industrial or food service industry with more accurate, exclusively progress glycosyl transfer Compound.

Summary of the invention

The purpose of the present invention is to provide novel UDP- glycosyl transferase and its applications.

In the first aspect of the present invention, the polypeptide of separation is provided, the polypeptide is selected from:

(a) there is SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 institute Show the polypeptide of amino acid sequence；

(b) in the space structure of the polypeptide of the amino acid sequence shown in SEQ ID NO:1, the 21st and the 121st bit amino Acid is constant, but is located near the 21st and the 121st amino acids position or occurs in substrate receptor binding pocket amino acid residue The polypeptide of variation；

(c) on the basis of the amino acid sequence shown in SEQ ID NO:1, the 16th, 82,83,86,90,119,124,126, 140,141,142,143,145,149,153,198,199,200,210,212,302,379,397 or 398 generation amino acid The polypeptide of variation；Preferably, P82S, T83I, I86R, I90L, F119I, A126V, A140M, L141M, S149G occurs, The polypeptide of L153F, Y212F or I302T variation；

(d) (a) or conservative variation's polypeptides (b) or (c).

In a preferred embodiment, the conservative variation's polypeptides are selected from:

(1) as (a) or (b) or (c) described in any polypeptide by one or more (such as 1-20, preferably 1-10； More preferably 1-5；More preferably 1-3) replacing, missing or adding for amino acid residue and formed, and have the function of polypeptide (a) Polypeptide；

(2) amino acid sequence of amino acid sequence and (a) or polypeptide (b) or (c) have 80% or more (preferably 85% with On；More preferably 90% or more；More preferably 95% or more；More preferably 99% or more) the phase same sex, and it is more with polypeptide (a) function Peptide；Or

(3) at (a) or (b) or (c), the N of the polypeptide or C-terminal add sequence label, or add signal in its N-terminal The polypeptide formed after peptide sequence；

Preferably, the 21st is histidine in the conservative variation's polypeptides of SEQ ID NO:1, the 121st is asparagine Acid.

In another preferred example, the conservative variation's polypeptides of the polypeptide of amino acid sequence shown in SEQ ID NO:1 include (but Be not limited to): (b) or (c) in, the position of alanine near the 21st hyte propylhomoserin does not occur for the mutation.

In another preferred example, in (b) or (c), the polypeptide be the 16th, 82,83,86,90,119,124,126, 140,141,142,143,145,149,153,198,199,200,210,212,302,379,397 or 398 generation amino acid The polypeptide of variation；Preferably, the polypeptide is that P82S, T83I, I86R, I90L, F119I, A126V, A140M occurs, The polypeptide of L141M, S149G, L153F, Y212F or I302T variation.

In another preferred example, the conservative variation's polypeptides of the polypeptide of amino acid sequence shown in SEQ ID NO:1 do not include: The polypeptide of amino acid variation occurs at the 16th, 198,199,397 or 398；Preferably there is no A16F, S198L, F199D, The variation of F397S or A398W.

In another aspect of this invention, the polynucleotides of separation are provided, it encodes any polypeptide in front.

In another aspect of this invention, a kind of carrier is provided, it contains the polynucleotides.

In another aspect of this invention, a kind of genetically engineered host cell is provided, it contain the carrier or its The polynucleotides are integrated in genome.

In another aspect of this invention, a kind of preparation method of any polypeptide in front is provided, this method includes: training Support the host cell；Collect the culture for containing the polypeptide.

In another preferred example, the method further include: it is any described that Claims 1 to 4 is isolated from culture Polypeptide.

In another aspect of this invention, the purposes of polypeptide is provided, wherein the polypeptide is with SEQ ID NO:2, SEQ The polypeptide or their conservative variation's polypeptides of amino acid sequence shown in ID NO:3, SEQ ID NO:4 or SEQ ID NO:5；With In shifting a upper glycosyl (Glc) on the compound with parent nucleus (I) structure, the compound such as formula (II) structure is formed:

Wherein, R is independently selected from H, OH, O- β-D-Glucose base, O- β-D-Glucose base-(2 → 1)-β-D-Glucose Base.

In a preferred embodiment, the compound with parent nucleus (I) structure includes: steviol, Ent-kauran Olefin(e) acid, steviol monoglycosides, steviol disaccharide glycosides；Or the compound of described formula (II) structure includes: the Portugal steviol -19-O- Grape sugar ester, Ent-kauran olefin(e) acid -19-O- glucose ester, sweetness agent classification glycosides, stevioside.

In another preferred example, wherein the polypeptide is have amino acid sequence shown in SEQ ID NO:1 polypeptide or Its conservative variation's polypeptides；For shifting a upper glycosyl (Glc) on the compound with parent nucleus (III) structure, formed such as The compound of formula (IV) structure:

Wherein, R ' is independently selected from H, O- β-D-Glucose base.

In another preferred example, the compound with parent nucleus (III) structure includes: steviol -19-O- glucose Ester, steviol；Or the compound of described formula (IV) structure includes: sweetness agent classification glycosides, steviol monoglycosides.

In another preferred example, the donor of the glycosyl is the compound containing glycosyl；Preferably, the donor packet It includes (but being not limited to): UDP-glucose, UDP- rhamnose, UDP- xylose；Or it is summarised as UDP- sugar.

In another aspect of this invention, a kind of composition for glycosyl transfer is provided, it contains the polypeptide or contains There are the host cell, its culture or pyrolysis product.

In another aspect of this invention, one kind is provided and shifts a upper glycosyl on the compound with parent nucleus (I) structure Method, which comprises with polypeptide, express the host cell, its culture or pyrolysis product of the polypeptide, or more containing this Glycosyl is transferred to the compound with parent nucleus (I) structure by the composition of peptide；

Wherein, R is independently selected from H, OH, O- β-D-Glucose base, O- β-D-Glucose base-(2 → 1)-β-D-Glucose Base；

Wherein, which has ammonia shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 The polypeptide of base acid sequence or their conservative variation's polypeptides.

In another aspect of this invention, one kind is provided and shifts a upper sugar on the compound with parent nucleus (III) structure The method of base, which comprises with polypeptide, express the host cell, its culture or pyrolysis product of the polypeptide, or containing should Glycosyl is transferred to the compound with parent nucleus (III) structure by the composition of polypeptide；

Wherein, R ' is independently selected from H, O- β-D-Glucose base；

Wherein, which has the polypeptide or their conservative variation's polypeptides of amino acid sequence shown in SEQ ID NO:1.

In a preferred embodiment, the donor of the glycosyl is the compound containing glycosyl；Preferably, the donor packet It includes (but being not limited to): UDP-glucose, UDP- rhamnose, UDP- xylose；Or it is summarised as UDP- sugar.

Other aspects of the invention are apparent to those skilled in the art due to this disclosure 's.

Detailed description of the invention

Figure 1A, with steviol (Steviol) be substrate, RsS19GT1, RsS19GT2, RsS19GT3 and RsS19GT4 are external The HPLC of enzymic catalytic reaction converted product is analyzed.

Figure 1B, with steviol (Steviol) be substrate, the LC-MS map of the external enzymic catalytic reaction converted product of RsS19GT1.

Fig. 1 C, with steviol (Steviol) be substrate, the LC-MS map of the external enzymic catalytic reaction converted product of RsS19GT2.

Fig. 2A, with steviol -19-O- glucose ester (steviol 19-O- β-D-glucoside) be substrate, RsS13GT The HPLC analysis of external enzymic catalytic reaction converted product.

Fig. 2 B, with steviol -19-O- glucose ester (steviol 19-O- β-D-glucoside) be substrate, RsS13GT The LC-MS map of external enzymic catalytic reaction converted product.

Fig. 3 A, the external enzymic catalytic reaction of RsS19GT1, RsS19GT2, RsS19GT3 and RsS19GT4 convert mapping-shell The HPLC of China fir olefin(e) acid (ent-kaur-16-en-19-oic acid) is analyzed.

Fig. 3 B, with Ent-kauran olefin(e) acid (ent-kaur-16-en-19-oic acid) for substrate, RsS19GT1 is external The LC-MS map of enzymic catalytic reaction conversion.

Fig. 3 C, with Ent-kauran olefin(e) acid (ent-kaur-16-en-19-oic acid) for substrate, RsS19GT2 is external The LC-MS map of enzymic catalytic reaction conversion.

Fig. 4 A, the external enzymic catalytic reaction of RsS19GT1, RsS19GT2, RsS19GT3 and RsS19GT4 convert steviol monosaccharide The HPLC of glycosides (steviolmonoside) is analyzed.

Fig. 4 B, with steviol monoglycosides (steviolmonoside) for substrate, RsS19GT1 external enzymic catalytic reaction conversion LC-MS map.

Fig. 4 C, with steviol monoglycosides (steviolmonoside) for substrate, RsS19GT2 external enzymic catalytic reaction conversion LC-MS map.

Fig. 5 A, the external enzymic catalytic reaction of RsS19GT1, RsS19GT2, RsS19GT3 and RsS19GT4 convert steviol disaccharide The HPLC of glycosides (steviolbioside) is analyzed.

Fig. 5 B, with steviol disaccharide glycosides (steviolbioside) for substrate, RsS19GT1 external enzymic catalytic reaction conversion LC-MS map.

Fig. 5 C, with steviol disaccharide glycosides (steviolbioside) for substrate, RsS19GT2 external enzymic catalytic reaction conversion LC-MS map.

Fig. 6 A, with steviol (steviol) for substrate, the HPLC analysis of RsS13GT external enzymic catalytic reaction conversion steviol.

Fig. 6 B, with steviol (steviol) for substrate, the LC-MS of RsS13GT external enzymic catalytic reaction conversion steviol Map.

The relative activity of each mutant of Fig. 7, RsS13GT (with RsS13GT wild type for 100%).

The relative activity (with RsS13GT wild type for 100%) of each mutant of Fig. 8, RsS13GT, measures 6 times, is averaged Value.

Specific embodiment

The present inventor filters out a system from this species of sweetness agent classification by large-scale screening and in-depth study Arrange the novel glycosylated uridine 5'-diphosphate of participation terpene (UDP)-glycosyl transferase.The glycosyl transferase can it is special and It is efficiently catalyzed the glycosylation of the C-4 carboxyl or C-13 hydroxyl of tetracyclic diterpene compound, to generate one kind glycosylated four Ring diterpene compound.

Active peptides, its encoding gene, carrier and host

As used herein, " isolated polypeptide " refer to the polypeptide substantially free of natural relative other albumen, Lipid, carbohydrate or other materials.Those skilled in the art can purify the polypeptide with the purified technology of protein of standard.Substantially Upper pure polypeptide can generate single master tape in non-reducing polyacrylamide gel.The purity of the polypeptide can also use amino Acid sequence is further analyzed.

Present invention discloses one group it is new, participate in the glycosylated uridine 5'-diphosphate of terpene (UDP)-glycosyl transferase.It is preferred that Ground, they are RsS13GT, RsS19GT1, RsS19GT2, RsS19GT3, RsS19GT4 and；They are respectively provided with SEQ ID NO: The polypeptide of amino acid sequence shown in 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5.The present invention It also include their conservative variation's polypeptides.

In the present invention, " conservative variation's polypeptides ", which refer to, is kept substantially the identical biology function of the polypeptide Energy or active polypeptide.

" conservative variation's polypeptides ", which can be (i), one or more conservative or non-conservative amino acid residues (preferably conservative amino acid) substituted polypeptide, and such substituted amino acid residue can be may not be by Genetic code encoding, or (ii) mature more with the polypeptide of substituent group, or (iii) in one or more amino acid residues Peptide and another compound (for example extending the compound of polypeptide half-life period, such as polyethylene glycol) fusion are formed by polypeptide, or (iv) additional amino acid sequence be fused to this polypeptide sequence and formed polypeptide (such as leader sequence or secretion sequence or be used to it is pure Change the sequence or proprotein sequence of this polypeptide, or the fusion protein with the formation of antigen I gG segment).According to the teaching of this article, this A little segments, derivative and analogue belong to scope known to those skilled in the art.

" conservative variation's polypeptides " may include (but being not limited to): one or more (usually 1-50, compared with Good ground 1-30, more preferably 1-20, most preferably 1-10) missing, insertion and/or the substitution of amino acid, and in C-terminal And/or N-terminal adds or lacks one or several (being more preferably within 5 within 20 or 10 within such as 50) ammonia Base acid.For example, in the art, when being substituted with similar nature or similar amino acid, not usually changing protein Function.For another example, the function of protein will not be changed by adding one or several amino acid generally also in C-terminal and/or N-terminal. The present invention also provides the analogs of the polypeptide.The difference of these analogs and natural polypeptides can be the difference on amino acid sequence It is different, it is also possible to not influence the difference on the modified forms of sequence, or have both at the same time.These polypeptides include natural or induction Genetic variant.Induction variant can be obtained by various technologies, such as be generated at random by radiating or being exposed to mutagens Mutagenesis can also pass through the technology of site-directed mutagenesis or other known molecular biology.Analog further includes having to be different from naturally The analog of the residue (such as D- amino acid) of l-amino acid, and with non-naturally occurring or synthesis amino acid (such as β, γ- Amino acid) analog.It should be understood that polypeptide of the invention is not limited to enumerated representative polypeptide.

Modification (not changing primary structure usually) form includes: the chemical derivative form such as acetyl of internal or external polypeptide Change or carboxylated.Modification further includes glycosylation, is carried out in the synthesis and processing of polypeptide or in further processing step such as those Glycosylation modified and generation polypeptide.This modification can carry out glycosylated enzyme (such as mammal by the way that polypeptide to be exposed to Glycosylase or deglycosylation enzyme) and complete.Modified forms further include with phosphorylated amino acid residue (such as phosphoric acid junket ammonia Acid, phosphoserine, phosphothreonine) sequence.It further include being modified to improve its anti-proteolytic properties or optimization The polypeptide of solubility property.

The aminoterminal or c-terminus of polypeptide of the invention can also be containing one or more polypeptide fragments, as protein tag. Any suitable label may be used to the present invention.For example, the label can be FLAG, HA, HA1, c-Myc, Poly- His, Poly-Arg, Strep-TagII, AU1, EE, T7,4A6, ε, B, gE and Ty1.These labels can be used for albumen into Row purifying.

In order to make the protein secretion expression (being such as secreted into extracellular) of translation, can also add in the aminoterminal of polypeptide of the invention In addition signal peptide sequence.Signal peptide can be cut out during polypeptide comes out from intracellular secretory.

On the basis of natural sequence, variation appropriate is carried out, the higher albumen of activity will be obtained.As of the invention The polypeptide (RsS13GT) of amino acid sequence shown in SEQ ID NO:1 is carried out rite-directed mutagenesis, by these single mutation by preferred embodiment Simultaneously heterogenous expression finds that the recombination mutation body protein of heterogenous expression can more efficiently be catalyzed Fourth Ring in Escherichia coli to body clone The glycosyl of the C-13 hydroxyl of diterpene-kind compound steviol -19-O- glucose ester (steviol19-O- β-D-glucoside) Change, to more efficiently generate sweetness agent classification glycosides (rubusoside).Therefore, in disclosed natural sequence On the basis of, the polypeptide obtain after variation appropriate is also contained in protection scope of the present invention.

Present invention further teaches a series of polypeptide variants for retaining bioactivity, comprising: the amino shown in SEQ ID NO:1 On the basis of acid sequence, the 16th, 82,83,86,90,119,124,126,140,141,142,143,145,149,153,198, 199, the polypeptide of 200,210,212,302,379,397 or 398 generation amino acid variations；Preferably, for the 82nd, 83,86, 90, the polypeptide of 119,126,140,141,149,153,212 or 302 generation amino acid variations.More specifically, comprising: in SEQ On the basis of amino acid sequence shown in ID NO:1, A16F, P82S, T83I, I86R, I90L, F119I, M124L, A126V occurs, A140M, L141M, I142Y, Y143W, I145L, S149G, L153F, S198L, F199D, C200W, F210L, Y212F, The polypeptide of I302T, N379G, F397S or A398W variation；Preferably, for P82S, T83I, I86R, I90L, F119I occurs, The polypeptide of A126V, A140M, L141M, S149G, L153F, Y212F or I302T.

Active peptides of the invention can be recombinant polypeptide, natural polypeptides, synthesis polypeptide.Polypeptide of the invention can be day The product or chemically synthesized product so purified, or use recombinant technique from protokaryon or eucaryon host (for example, bacterium, ferment Female, higher plant) in generate.According to host used in recombinant production scheme, polypeptide of the invention can be it is glycosylated, or can To be nonglycosylated.Polypeptide of the invention may also include or not include the methionine residues of starting.

Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.Term " coding polypeptide Polynucleotides " can be the polynucleotides including encoding this polypeptide, be also possible to further include additional code and/or non-coding sequence The polynucleotides of column.

The invention further relates to the variant of above-mentioned polynucleotides, coding has the more of identical amino acid sequence with the present invention The segment of peptide or polypeptide, analogs and derivatives.The variant of this polynucleotides can be the allelic variant naturally occurred or The variant that non-natural occurs.These nucleotide variants include substitution variants, Deletion variants and insertion variant.Such as this Known to field, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution, Missing or insertion, but not from substantially change its encode polypeptide function.

The invention further relates to hybridizing with above-mentioned sequence and having at least 50% between two sequences, preferably at least 70%, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under stringent condition (or stringent condition) with The interfertile polynucleotides of polynucleotides of the present invention.In the present invention, " stringent condition " refers to: (1) strong in lower ion Hybridization and elution under degree and higher temperature, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant when (2) hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or (3) are only identical between two sequences Property at least just hybridizes at 90% or more, more preferably 95% or more.

The invention further relates to the nucleic acid fragments hybridized with above-mentioned sequence.As used herein, the length of " nucleic acid fragment " is extremely Contain 15 nucleotide, preferably at least 30 nucleotide, more preferably at least 50 nucleotide, preferably at least 100 nucleosides less It is more than acid.

Polypeptide and polynucleotides in the present invention preferably provide in a separate form, are more preferably purified to homogeneous.

Nucleotide full length sequence of the invention or its segment can usually use PCR amplification method, recombination method or artificial synthesized Method obtains.Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method. In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.Chemical synthesis can also be passed through Mutation is introduced into protein sequence of the present invention.

It is optimized for obtaining gene of the invention using round pcr DNA amplification/RNA method.Especially it is difficult from text When obtaining the cDNA of overall length in library, RACE method (end RACE-cDNA rapid amplification) preferably is used, the primer for PCR It can be properly selected according to the sequence information of invention disclosed herein, and available conventional method synthesis.Conventional method can be used The DNA/RNA segment of amplification is such as separated and purified by gel electrophoresis.

The present invention also relates to the carriers comprising polynucleotides of the invention, and with carrier of the invention or peptide coding sequence Arrange genetically engineered host cell, and the method for generating polypeptide of the present invention through recombinant technique.

By the recombinant dna technology of routine, it can be used to express or produce weight using polynucleotide sequence of the invention RsS19GT1, RsS19GT2, RsS19GT3, RsS19GT4 and the RsS13GT polypeptide of group.In general there are following steps:

(1) (is contained with coding RsS19GT1, RsS19GT2, RsS19GT3, RsS19GT4 and RsS13GT polypeptide of the invention Its conservative variation's polypeptides) polynucleotides, or with containing the polynucleotide recombinant expression carrier converted or transduceed it is suitable Host cell；

(2) host cell that is cultivated in suitable culture medium；

(3) be separated from culture medium or cell, protein purification.

In the present invention, the polynucleotides sequence of RsS19GT1, RsS19GT2, RsS19GT3, RsS19GT4 or RsS13GT are encoded Column can be plugged into recombinant expression carrier.Term " recombinant expression carrier " refers to bacterial plasmid well known in the art, bacteriophage, yeast Plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus or other carriers.As long as can be in host It replicates and stablizes in vivo, any plasmid and carrier can be used.One important feature of expression vector is to usually contain to replicate Point, promoter, marker gene and translation control element.

Method well-known to those having ordinary skill in the art can be used to construct containing RsS19GT1, RsS19GT2, RsS19GT3, The expression vector of the DNA sequences encoding of RsS19GT4 and RsS13GT and suitable transcription/translation control signal.These method packets Include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..The DNA sequence dna can be effectively connected to expression and carry In appropriate promoter in body, to instruct mRNA to synthesize.In addition, expression vector preferably includes one or more selected markers Gene, to provide the phenotypic character for selecting the host cell of conversion.

Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for converting suitable When host cell, allow it to expression protein.Host cell can be prokaryotic cell, such as bacterial cell；Or it is low Eukaryocyte, such as yeast cells；Or higher eucaryotic cells, such as mammalian cell.Representative example has: Escherichia coli, chain Mould category, hay bacillus；The bacterial cell of salmonella typhimurium；Fungal cell's such as yeast, plant cell, glossy ganoderma cell；Fruit The insect cell of fly S2 or Sf9；CHO, COS, 293 cells or zooblast of Bowes melanoma cells etc..

In method of the invention, the recombinant polypeptide can express or be secreted into cell in the cell or on cell membrane Outside.If desired, the egg of recombination can be separated by various separation methods and be purified using its physics, chemical and other characteristics It is white.These methods are well-known to those skilled in the art.The example of these methods includes but is not limited to: at conventional renaturation Reason breaks bacterium, super processing, ultracentrifugation, sieve chromatography (gel mistake with protein precipitant processing (salting-out method), centrifugation, infiltration Filter), adsorption chromatography, ion-exchange chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods Combination.

As preferred embodiment of the invention, the host cell is prokaryotic cell；Preferably, the prokaryotic host cell Including but not limited to: Escherichia coli.In the preferred embodiment, by glycosyltransferase gene RsS19GT1, Simultaneously heterogenous expression finds heterogenous expression in Escherichia coli to RsS19GT2, RsS19GT3, RsS19GT4 and RsS13GT clone Recombinant protein can specifically and/or efficiently be catalyzed tetracyclic diterpene compound Kaurenic acid, steviol and its derivative The glycosylation of C-4 carboxyl or C-13 hydroxyl, to generate the glycosylated tetracyclic diterpenes such as sweetness agent classification glycosides (rubusoside) Compound.

In the preferred embodiment, will carry the novel glycosyl transferase RsS19GT1 of sweetness agent classification and The plasmid of key function gene needed for the plasmid and carrying building steviol (steviol) skeleton of RsS13GT, corotation is in large intestine bar In bacterium, colibacillus engineering is obtained.By the colibacillus engineering of building, Low- temperature culture ferments in TB culture medium, obtains Fermentation liquid.Fermentation liquid centrifugation, obtains supernatant and thallus.Thallus is suspended with water, uses extracting n-butyl alcohol after crushing；Supernatant is used Extracting n-butyl alcohol merges butanol extraction liquid.It is redissolved after butanol extraction liquid is evaporated with methanol, carries out HPLC analysis, obtained Required product.

Using

RsS19GT1, RsS19GT2, RsS19GT3, RsS19GT4 or RsS13GT polypeptide of the invention or theirs is conservative Property Variant polypeptides, can be applied to the glycosyl of C-4 carboxyl or C-13 hydroxyl special and that be efficiently catalyzed tetracyclic diterpene compound Change, to generate a kind of glycosylated tetracyclic diterpene compound.

As a kind of preferred embodiment of the invention, the polypeptide for playing catalytic activity is RsS19GT1, RsS19GT2, RsS19GT3 or RsS19GT4, and following reactions can be catalyzed:

Herein, the tetracyclic diterpene compound is such as, but not limited to steviol, Ent-kauran olefin(e) acid, steviol Monoglycosides, steviol disaccharide glycosides and its derivative；It is respectively steviol -19-O- glucose plus the product after a glycosyl Ester, Ent-kauran olefin(e) acid -19- glucose ester, sweetness agent classification glycosides, stevioside.

As a kind of preferred embodiment of the invention, the polypeptide for playing catalytic activity is RsS13GT, and can be catalyzed following Reaction:

Herein, the tetracyclic diterpene compound is such as, but not limited to steviol -19-O- glucose ester, steviol；Its In addition the compound that the product after a glycosyl is respectively described formula (IV) structure includes: sweetness agent classification glycosides, steviol list Glucosides.

In use, especially in industrialized production, RsS19GT1, RsS19GT2, RsS19GT3 of the invention, RsS19GT4 and RsS13GT polypeptide or their conservative variation's polypeptides also can be fixed on other solid phase carriers, obtain The enzyme of immobilization is applied to carry out vitro reactions with substrate.The solid phase carrier is, for example, microballoon made of some inorganic matters, Tubular body etc..The preparation method of immobilised enzymes has physical method and chemical method two major classes.Physical method includes physisorphtion, embedding Method etc..Chemical method includes combined techniques, cross-linking method.Combined techniques are divided into ions binding method and covalent coupling method again.Above-mentioned immobilization The method of enzyme can be applied in the present invention.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or According to the normal condition proposed by manufacturer.

Material

Sweetness agent classification (Rubus suavissimus S.Lee) plant leaf blade is derived from South China Botanical Garden Chinese Academy of Sciences.

Oligonucleolide primers are purchased from raw industry science skill (Sangon Biotech) Co., Ltd.

Sequence analysis commission Shanghai Mei Ji biological medicine Co., Ltd.

AxyPrep total serum IgE Miniprep Kit, polymerase chain reaction (PCR) plastic recovery kit, plasmid extraction examination Agent box is U.S.'s Axygen product.

PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time) polymerase Kit, polymerase chain reaction (PCR) high fidelity enzyme PrimeSTAR Max DNA Polymerase are that the precious biology of Japan is public Take charge of (TAKARA) product.

Restriction enzyme is NEB product.

Escherichia coli DH10B, Rosetta (DE3) bacterial strain and pET21a, pET28a carrier are used for gene cloning and albumen table It reaches.

Standard items compound steviol (steviol), steviol disaccharide glycosides (steviolbioside) are purchased from Shanghai source leaf Biotechnology Co., Ltd, sweetness agent classification glycosides (rubusoside) are purchased from Nanjing Guang Run biological products Co., Ltd.

UDP glucose is purchased from biological Co., Ltd safe in Beijing.

Other reagents are that domestic analysis is pure or chromatography pure reagent, are purchased from Sinopharm Chemical Reagent Co., Ltd..Chromatography point From using reverse phase silica gel (ODS-A-HG, 12nm, S-50 μm, YMC Co., Ltd., Tokyo, Japan).

PCR uses Arktik Thermal Cycler (Thermo Fisher Scientific)；Constant temperature incubation uses ZXGP-A2050 constant incubator (intelligence city) and ZWY-211G constant temperature culture oscillator (intelligence city)；Centrifugation uses 5418R high speed cold Jelly formula centrifuge and 5418 compact centrifuges (Eppendorf).Vacuum concentration uses Concentrator plus concentrating instrument (Eppendorf)；OD₆₀₀It uses the detection of UV-1200 ultraviolet-uisible spectrophotometer (U.S. spectrum in Shanghai reaches Instrument Ltd.).Rotation Turn vapo(u)rization system by 10 digital Rotary Evaporators (IKA) of IKA RV and MZ 2C NT chemical membrane pump, CVC3000 vacuum Controller (vacuubrand) composition.High performance liquid chromatography uses 3000 liquid chromatographic system of Dionex UltiMate (Thermo Fisher Scientific).NMR spectrum is obtained by the measurement of Bruker DRX 500；High resolution mass spectrum by Thermo Fisher Scientific electrostatic field orbit trap combination mass spectrum Q Exactive is measured.

Embodiment 1, novel uridine 5'-diphosphate (UDP)-glycosyl transferase

1, the preparation of sweetness agent classification leaf cDNA

Sweetness agent classification mature leaf is taken, clear water cleans up surface blot, gently wipes blade surface with blotting paper It is water stain, it weighs spare.It is wrapped up using masking foil, liquid nitrogen flash freezer, -80 DEG C of cryopreservations are spare.With reference to the AxyPrep of Axygen Total serum IgE Miniprep Kit specification prepares blade total serum IgE.With reference to the PrimeScript RT reagent Kit of TaKaRa With gDNA Eraser (Perfect Real Time) reverse transcription reagent box specification, reverse transcription RNA prepare cDNA.

2, RsS19GT1, RsS19GT2, RsS19GT3, RsS19GT4 and RsS13GT Gene clone and expression plasmid construction

It designs primer shown in table 1 and introduces suitable restriction enzyme site in upstream and downstream.50 μ L of PCR amplification system: PrimeSTAR Max Premix, 25 μ L；0.2~0.3 μM of 2 final concentration of Primer 1/Primer；cDNA<200ng；Residual body Product is supplied with sterile purified water.

PCR reaction condition: 98 DEG C of initial denaturation 2min, then 98 DEG C of denaturation 10s, 55 DEG C of annealing 15s, 72 DEG C of extension 20s, 28 A circulation, 72 DEG C of extension 10min, agarose electrophoresis detection, amplification obtain the segment of about 1.5kb, after purification with the digestion introduced Site digestion.

Digestion post-fragment is connected into pET21a the and pET28a carrier of identical enzymic digestion, and connection product converts Escherichia coli DH10B competent cell, introduces the verifying of restriction enzyme site double digestion and gene sequencing when extraction plasmid is constructed, screening is recombinated Plasmid.

Table 1, primer

3, the expression of RsS19GT1, RsS19GT2, RsS19GT3, RsS19GT4 and RsS13GT

By in the competent cell of recombinant plasmid transformed to Escherichia coli Rosetta (DE3), (chlorine is mould for LB solid medium Element 34 μ g/mL, 100 μ g/mL of ampicillin) 37 DEG C of overnight incubations.Picking is individually cloned into 2mL LB liquid medium, and (chlorine is mould Element 34 μ g/mL, 100 μ g/mL of ampicillin), the bacterium solution being incubated overnight is transferred into new LB liquid resistance culture base 37 DEG C, 250r/min is cultivated to OD₆₀₀=0.3~0.5, water-bath is cooled to 16 DEG C or so, and inducer IPTG is then added to final concentration 0.1mM goes to 16 DEG C of low temperature induction cultures, continues to cultivate 20h under the conditions of shaking speed 180r/min.Carrying is not connected into external Rosetta (DE3) recombinant bacterial strain of the pET21a and pET28a empty plasmid of gene is same as above as blank control, culture operation.Table After reaching, expression is examined using SDS-PAGE.Bacterium solution is centrifuged (12000r/min, 1min), discards supernatant, with isometric Sterile water is resuspended, and sets ultrasonication on ice and extremely clarifies, crude enzyme liquid refrigerated centrifuge (10000r/min, 10min, 4 DEG C) after ultrasound, Supernatant is taken to precipitate respectively, SDS-PAGE verifies protein expression situation.

Embodiment 2, the external Function Identification of RsS19GT1, RsS19GT2, RsS19GT3, RsS19GT4 and RsS13GT

The monoclonal glycerol stock saved transfer to 2mL LB liquid medium (chloramphenicol 34 μ g/mL, 100 μ of ampicillin G/mL in), 37 DEG C, 250rpm be incubated overnight, by 1% (v/v) bacterium solution that is incubated overnight of switching to new 50mL LB Liquid Culture In base, 37 DEG C, 250rpm cultivates to OD₆₀₀=0.3~0.5, IPTG to final concentration 0.1mM is added, 16 DEG C, 180rpm induction is trained Support 20h.Rosetta (DE3) recombinant bacterial strain of pET21a and pET28a empty plasmid is carried as blank control, operation is same as above.From The heart receives bacterium, breaks bacterium buffer solution B uffer A (20mM Tris-HCl, pH 8.0 using 5mL；100mM NaCl) it is resuspended, it is added DNase is to final concentration of 5 μ g/mL, Mg²⁺The final concentration of 1mM of final concentration of 2mM, protease inhibitors PMSF, is incubated on ice Ultrasonication after 30min；It is centrifuged (10000rpm, 30min, 4 DEG C), by 0.2 μm of membrane filtration, obtains supernatant crude enzyme liquid.

Vitro enzyme catalytic determination system is 500 μ L, and reaction system includes: glycosyl acceptor (steviol, the steviol of 0.25mM Monoglycosides, steviol disaccharide glycosides, Ent-kauran olefin(e) acid, steviol -19-O- glucose ester), the UDP glucose of 1.5mM, 10mM MgCl₂, 200 μ L supernatant crude enzyme liquids supply volume to 500 μ L with Buffer A.37 DEG C of reaction 2h.To after reaction, Add isometric ethyl acetate (when using steviol and Kaurenic acid as substrate) or n-butanol (when other substrates) to terminate reaction, uses Isometric ethyl acetate or extracting n-butyl alcohol 3 times, by extract for 3 times it is organic mix, be centrifuged and be evaporated in vacuum concentration instrument, Then it is redissolved using 500 μ L methanol or methanol-water (50/50, v/v), carries out product analysis using HPLC.HPLC testing conditions: Chromatographic column: reverse phase C18 column [TSKge1 ODS-1V ], Detection wavelength 205nm；Mobile phase: second Nitrile-aqueous mixtures gradient elution, ratio is raised to 80:20, flow velocity 1mL/min by 30:70 in 0 to 25min.

RsS19GT1, RsS19GT2, RsS19GT3 and RsS19GT4 participate in the HPLC testing result for the product that reaction obtains Such as Figure 1A.According to Figure 1A, RsS19GT1, RsS19GT2, RsS19GT3 and RsS19GT4 can be by substrate steviol (Steviol) It is converted into its glycation product.It is C that the glycation product, which detects confirmation molecular formula through negative ion mode high resolution mass spectrum,₂₆H₄₀O₈, Nuclear magnetic resonance spectroscopy and carbon spectrum display dammara ene-type tetracyclic diterpene parent nucleus characteristic signal and glycosyl characteristic signal, in two-dimentional nuclear-magnetism Confirm that 19 carboxyls of 1 ' position terminal hydrogen of compound glucose and steviol parent nucleus have in Heteronuclear multiple bond Correlated Spectroscopy (HMBC) Long-range relevant peaks.

With steviol (Steviol) for substrate, it is converted steviol -19-O- glucose ester by RsS19GT1 The LC-MS map such as Figure 1B of (steviol 19-O- β-D-glucoside).

With steviol (Steviol) for substrate, it is converted steviol -19-O- glucose ester by RsS19GT2 LC-MS map such as Fig. 1 C of (steviol 19-O- β-D-glucoside).

Hence, it can be determined that glycation product obtained is steviol -19-O- glucose ester (steviol-19-O- β - D-glucoside), the reaction equation of the reaction of generation is as follows:

RsS13GT participates in the HPLC testing result such as Fig. 2A, LC-MS map such as Fig. 2 B for the product that reaction obtains.According to figure 2, RsS13GT can convert substrate steviol -19-O- glucose ester (steviol19-O- β-D-glucoside) to its sugar Base product.It is C that the product, which detects confirmation molecular formula through negative ion mode high resolution mass spectrum,₃₂H₅₀O₁₃, with sweetness agent classification glycosides (rubusoside) molecular formula is consistent, and HPLC retention time is also consistent with sweetness agent classification glycosides standard items, therefore identifying should Product is sweetness agent classification glycosides.

The Substratspezifitaet research of embodiment 3, RsS19GT1, RsS19GT2, RsS19GT3, RsS19GT4 and RsS13GT

1、RsS19GT1、RsS19GT2、RsS19GT3、RsS19GT4

RsS19GT1, RsS19GT2, RsS19GT3 and RsS19GT4 can not only identify steviol (Steviol), simultaneously For the analogue Ent-kauran olefin(e) acid (ent-kaur-16-en-19-oic acid) of steviol, steviol monosaccharide Glycosides (steviolmonoside), steviol disaccharide glycosides (steviolbioside) have 19 glycosylation catalytic activity, respectively It generates Ent-kauran olefin(e) acid -19- glucose ester (β-D-glucosyl-ent-kaur-16-en-19-oate), sweetleaf stirrup Sub- glycosides (rubusoside) and stevioside (stevioside).HPLC analysis chart such as Fig. 3 A, 4A, 5A.

With Ent-kauran olefin(e) acid (ent-kaur-16-en-19-oic acid) for substrate, RsS19GT1 converts it For the LC-MS map of Ent-kauran olefin(e) acid -19- glucose ester (β-D-glucosyl-ent-kaur-16-en-19-oate) Such as Fig. 3 B.The LC-MS map such as Fig. 3 C accordingly converted with RsS19GT2.

With steviol monoglycosides (steviolmonoside) for substrate, it is converted sweetness agent classification glycosides by RsS19GT1 (rubusoside) LC-MS map such as Fig. 4 B.The LC-MS map such as Fig. 4 C accordingly converted with RsS19GT2.

With steviol disaccharide glycosides (steviolbioside) for substrate, it is converted stevioside by RsS19GT1 (stevioside) LC-MS map such as Fig. 5 B.The LC-MS map such as Fig. 5 C accordingly converted with RsS19GT2.

The substrate parent nucleus and its product structure general formula of RsS19GT1, RsS19GT2, RsS19GT3 and RsS19GT4 identification are such as Under:

2、RsS13GT

RsS13GT can not only identify steviol -19-O- glucose ester (steviol 19-O- β-D-glucoside), Also there is 13 glycosylation catalytic activity simultaneously for steviol, generate steviol monoglycosides (steviolmonoside). HPLC analysis chart such as Fig. 6 A, LC-MS map such as Fig. 6 B.

The substrate of RsS13GT identification and its reaction formula of enzyme reaction catalysate are as follows:

Embodiment 4, uridine 5'-diphosphate (UDP)-glycosyl transferase RsS13GT transformation

1, the selection in mutational site

21 hyte propylhomoserin (by taking RsS13GT as an example) of uridine 5'-diphosphate (UDP)-glycosyl transferase and 121 asparagines Sour (by taking RsS13GT as an example) is its catalytic center, and the amino acid residue around this catalytic center may influence the knot of substrate and enzyme It closes.The present inventor selects around H21 and D121Residue in distance range is mutated.The present inventor predicts may be with The receptor pocket of substrate structure selects the residue that may be interacted with substrate to be mutated.

2, the building of the expression vector of RsS13GT gene mutation body

Primer shown in table 2 is designed for introduction to rite-directed mutagenesis.

Table 2, the primer constructed for RsS13GT gene mutation body

PCR amplification system 50 μ L:PrimeSTAR Max Premix, 25 μ L；2 final concentration of Primer 1/Primer, 0.2 μ M；RsS13GT wild type expression vector plasmid 10ng；Residual volume is supplied with sterile purified water.

PCR reaction condition: 98 DEG C of initial denaturation 2min, then 98 DEG C of denaturation 10s, 55 DEG C of annealing 5s, 72 DEG C of extension 3min, 30 A circulation, agarose electrophoresis detection, amplification obtain the segment of about 6kb.

PCR reaction product converts Escherichia coli DMT competent cell after DpnI is handled, and extracts plasmid through gene sequencing, Screening obtains directed mutants.

3, the expression of RsS13GT mutant

Recombinant mutant plasmid is transformed into the competent cell of e. coli jm109 (DE3), LB solid medium (card receive 50 μ g/mL of mycin, 20 μ g/mL of acidum nalidixicum) 37 DEG C of overnight incubations.Picking is individually cloned into 2mL LB liquid medium (card Receive 50 μ g/mL of mycin, 20 μ g/mL of acidum nalidixicum), the bacterium solution being incubated overnight is transferred into new LB liquid resistance culture base 37 DEG C, 250r/min is cultivated to OD₆₀₀=0.3~0.5, water-bath is cooled to 16 DEG C or so, and inducer IPTG is then added to final concentration 0.1mM goes to 16 DEG C of low temperature induction cultures, continues to cultivate 20h under the conditions of shaking speed 200r/min.Carry wild type JM109 (DE3) recombinant bacterial strain of RsS13GT expression vector is same as above as control, culture operation.After expression, use SDS-PAGE examines expression.Bacterium solution is centrifuged (6000r/min, 5min), discards supernatant, with 1/10 volume Buffer A (20mM Tris-HCl, pH 8.0；100mM NaCl) it is resuspended, DNase is added to final concentration of 5 μ g/mL, Mg²⁺It is final concentration of The final concentration of 1mM of 2mM, protease inhibitors PMSF is incubated for ultrasonication after 30min on ice；It is centrifuged (10000rpm, 30min, 4 DEG C), take supernatant to precipitate respectively, SDS-PAGE verifies protein expression situation.Supernatant is by 0.2 μm of membrane filtration, in acquisition Clear crude enzyme liquid.

Embodiment 5, the external Function Identification 1 of RsS13GT mutant

Vitro enzyme catalytic determination system is 500 μ L, and reaction system includes: the steviol -19-O- glucose ester of 0.25mM (steviol 19-O- β-D-glucoside), the UDP glucose of 1.5mM, 10mM MgCl₂, the 100 thick enzymes of μ L mutant supernatant Liquid supplies volume to 500 μ L with Buffer A.37 DEG C of reaction 2h.To after reaction, add isometric n-butanol to terminate reaction, With isometric extracting n-butyl alcohol 3 times, 3 the organic of extraction are mixed, is centrifuged and is evaporated in vacuum concentration instrument, then use 500 μ L methanol or methanol-water (50/50, v/v) redissolve, and carry out product analysis using HPLC.HPLC testing conditions: chromatographic column: anti- PhaseDetection wavelength is 205nm；Mobile phase: second Nitrile-aqueous mixtures gradient elution, ratio is raised to 60:40, flow velocity 1mL/min by 20:80 in 0 to 20min.

It is detected through HPLC, mutant RsS13GT F119I, RsS13GT L141M, RsS13GT Y143W, RsS13GT I145L, RsS13GT N379G and RsS13GT FA398SW can be by substrate steviol -19-O- glucose ester (steviol 19-O- β-D-glucoside) its 13 glycation product sweetness agent classification glycosides (rubusoside) are converted into, HPLC retains Time is also consistent with sweetness agent classification glycosides standard items.Compared with wild type RsS13GT, the relative activity of these mutant is shown such as Fig. 7.Mutant RsS13GT F119I (relative activity 167%), RsS13GT M124L (relative activity 213%), RsS13GT L141M (relative activity 155%) and RsS13GT I142Y (relative activity 112%) is opposite compared with the activity of wild type RsS13GT to be mentioned It is high.

Should the result shows that, near the space structure of D121There is activity after amino acid residue mutation in distance range Larger raising.In addition, for the transformation of alanine (A) may make activity decline to a great extent (mutant A16F, FA39398SW)。

Embodiment 6, the external Function Identification 2 of RsS13GT mutant

To RsS13GT mutant A16F, P82S, T83I, I86R, I90L, F119I, M124L, A126V, A140M, L141M, I142Y, Y143W, I145L, S149G, L153F, S198L, F199D, C200W, F210L, Y212F, I302T, N379G, F397S, A398W.

Substantially with embodiment 6, difference is to carry out 6 times for vitro enzyme catalytic determination system, HPLC detection architecture and method HPLC measuring, takes statistical average.

The results show that mutant RsS13GT P82S, RsS13GT T83I, RsS13GT I86R, RsS13GT I90L, RsS13GT F119I, RsS13GT M124L, RsS13GT A126V, RsS13GT A140M, RsS13GT L141M, RsS13GT I142Y, RsS13GT Y143W, RsS13GT I145L, RsS13GT S149G, RsS13GT L153F, RsS13GT S198L, RsS13GT C200W, RsS13GT F210L, RsS13GT Y212F, RsS13GT I302T, RsS13GT N379G, RsS13GT F397S and RsS13GT A398W can be by substrate steviol -19-O- glucose ester (steviol 19-O- β-D- Glucoside) be converted into its 13 glycation product sweetness agent classification glycosides (rubusoside), HPLC retention time also with sweet tea Leaf raspberry glycosides standard items are consistent.Compared with wild type RsS13GT, the relative activity of these mutant shows such as Fig. 8.Mutant RsS13GT P82S (relative activity 240%), RsS13GT T83I (relative activity 231%), RsS13GT I86R (relative activity 197%), RsS13GT I90L (relative activity 113%), RsS13GT F119I (relative activity 192%), RsS13GT A126V (relative activity 247%), RsS13GT A140M (relative activity 250%), RsS13GT L141M (relative activity 224%), RsS13GT S149G (relative activity 175%), RsS13GT L153F (relative activity 230%), RsS13GT Y212F are (opposite Activity is 382%) and RsS13GT I302T (relative activity 134%) is opposite compared with the activity of wild type RsS13GT improves.

Should the result shows that, near the space structure of D121Amino acid residue (120-140) in distance range Activity improves a lot after mutation, 80-90, may influence after the 149th, 153,212 and 302 mutation mutual with substrate Effect, so that the larger raising of activity.In addition, activity may be made for the transformation of the alanine (A) near the 21st hyte propylhomoserin Decline to a great extent (mutant A16F, A398W).

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>Shanghai Inst. of Life Science, CAS

<120>novel UDP- glycosyl transferase and its application

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Tyr Ile Cys Phe Gly Ser Ile Asn Ser Phe Ser Asp Cys Glu Leu Leu

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Asp Gly Phe Asp Gly Gly Val Lys Pro Asp Asp Pro Asn His Ile Met

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Ala Ser Ala Phe Leu Cys Ile Gln Ser Thr Thr Ser Phe Ala Leu Cys

130 135 140

Tyr His Tyr Phe Lys Asp Cys Tyr Lys Asp Gln Ser Thr Leu Pro Phe

145 150 155 160

Pro Ser Cys Ile Thr Ile Asp Gly Met Pro Pro Phe Ala Pro Glu Ala

165 170 175

Leu Pro Ser Tyr Leu Leu Pro Thr Ser Pro His Val Ser Ile Leu Pro

180 185 190

Thr Phe Gln Glu His Tyr Gln Ile Leu Glu Arg Asp Pro Asn Ser Cys

195 200 205

Val Leu Leu Asn Thr Phe Asp Gly Leu Glu Glu Ala Ala Ile Arg Asp

210 215 220

Met Arg Gly His Met Asn Leu Ile Thr Val Gly Pro Leu Phe Arg Ser

225 230 235 240

Asn Asp Ala Glu Val Arg Cys Asp Leu Phe Asp Arg Ser Gly Asp Asp

245 250 255

Tyr Leu Gln Trp Leu Asp Ser Lys Ala Asp Ser Ser Val Val Tyr Val

260 265 270

Ser Phe Gly Ser Met Val Val Leu Lys Ser Gly Gln Ile Glu Glu Ile

275 280 285

Leu His Gly Leu Val Asp Ser Gly Leu Pro Val Leu Trp Val Ile Arg

290 295 300

Lys Ser Gly Asn Glu Gly Asp Glu Glu Thr Leu Gln Asn Leu Ile Asn

305 310 315 320

Ser Thr Leu Lys Lys Glu Gln Gly Leu Ile Val Pro Trp Cys Ser Gln

325 330 335

Val Glu Val Leu Ser His Lys Ser Ile Gly Cys Phe Val Thr His Cys

340 345 350

Gly Trp Asn Ser Thr Ile Glu Ser Leu Ala Ala Gly Val Pro Ile Val

355 360 365

Gly Cys Pro His Phe Ser Asp Gln Asn Thr Asn Ala Lys Leu Val Glu

370 375 380

Glu Leu Trp Gly Thr Gly Val Arg Ala Arg Val Asn Ala Glu Gly Leu

385 390 395 400

Phe Glu His Asp Glu Ile Lys Arg Cys Leu Glu Met Val Ile Gly Asp

405 410 415

Gly Gln Thr Gly Glu Glu Ile Arg Arg Asn Ala Gln Lys Trp Lys Gly

420 425 430

Leu Ala Met Glu Ala Val Lys Glu Gly Gly Ala Ser Asn Asp Asn Leu

435 440 445

Met Asn Phe Val Ile Arg Leu Ser

450 455

<210> 5

<211> 489

<212> PRT

<213>sweetness agent classification (Rubus suavissimus S. Lee)

<400> 5

Met Ala Ser Gln Glu Cys Gln Leu His Phe Val Leu Phe Pro Phe Met

1 5 10 15

Ala Gln Gly His Met Ile Pro Met Ile Asp Ile Ala Arg Leu Leu Ala

20 25 30

Glu Arg Gly Leu Ile Ile Thr Ile Val Thr Thr Pro His Asn Ala Ser

35 40 45

Arg Phe Asp Lys Val Leu Ala Arg Ala Arg Glu Ser Gly Leu Gln Ile

50 55 60

Arg Leu Ile Gln Leu Lys Phe Pro Cys Glu Glu Ala Gly Leu Pro Arg

65 70 75 80

Gly Cys Glu Asn Val Asp Met Met Pro Ser Thr Asn Leu Ala Ser Ser

85 90 95

Phe Phe Asn Gly Thr Lys Gly Leu Gln Glu Pro Val Glu Lys Leu Phe

100 105 110

Glu Glu Leu Thr Pro Lys Pro Ser Cys Ile Ile Ser Asp Met Cys Leu

115 120 125

Pro Trp Thr Ile Asn Ile Ser His Met Phe Lys Ile Pro Arg Ile Ser

130 135 140

Phe Ser Gly Thr Cys Cys Phe Leu Leu Val Cys Phe Gly Asn Ala Cys

145 150 155 160

Ile Ser Lys Val Val Pro Asp Ile Thr Ser Glu Thr Glu Tyr Phe Val

165 170 175

Leu Pro Asp Leu Pro Asp Arg Ile Glu Val Thr Lys Ala Gln Leu Pro

180 185 190

Ala Ala Ile Ala Pro Asn Met Thr Asp Phe Thr Glu Thr Leu Arg Ala

195 200 205

Ala Glu Leu Ala Ser Tyr Gly Val Ile Met Asn Ser Phe Glu Glu Leu

210 215 220

Glu Pro Ala Tyr Val Glu Glu Tyr Lys Lys Val Lys Lys Asp Lys Leu

225 230 235 240

Trp Cys Ile Gly Pro Ala Ser Leu Cys Asn Lys Asp Glu Leu Asp Lys

245 250 255

Ala Gln Arg Gly Asn Lys Ala Ser Val Asp Glu His His Cys Leu Lys

260 265 270

Trp Leu Asp Ser Trp Glu Pro Ser Ser Val Leu Tyr Ala Cys Phe Gly

275 280 285

Ser Leu Cys Asn Leu Ile Pro Ala Gln Leu Ile Glu Leu Gly Leu Gly

290 295 300

Leu Glu Ala Ser Asn Lys Pro Phe Ile Trp Val Val Arg Gly Ser Ser

305 310 315 320

Gln Leu Glu Glu Leu Asp Lys Trp Ile Ala Glu Asn Arg Phe Glu Glu

325 330 335

Arg Thr Lys Gly Arg Ser Leu Leu Ile Arg Gly Trp Ala Pro Gln Thr

340 345 350

Leu Ile Leu Ser His Pro Ala Val Gly Gly Phe Leu Thr His Cys Gly

355 360 365

Trp Asn Ser Thr Leu Glu Gly Ile Cys Ala Gly Val Pro Leu Ile Thr

370 375 380

Trp Pro Leu Phe Gly Asp Gln Phe Leu Asn Glu Lys Leu Val Glu Gln

385 390 395 400

Ile Leu Lys Ile Ala Val Arg Val Gly Val Glu Tyr Pro Met Lys Trp

405 410 415

Gly Glu Glu Glu Asn Ile Gly Val Leu Val Lys Lys Glu Asn Val Met

420 425 430

Glu Ala Ile Asp Lys Leu Met Asp Gly Glu Glu Ser Leu Ala Arg Arg

435 440 445

Asp Arg Ala Arg Glu Leu Ala Lys Met Ala Lys Arg Ala Val Glu Glu

450 455 460

Gly Gly Ser Ser His Leu Asn Ile Glu Leu Leu Ile Lys Asp Ile Met

465 470 475 480

Gln Gln Gly Asn Cys Thr Lys Thr Asn

485

<210> 6

<211> 29

<212> DNA

<213>primer (Primer)

<400> 6

ggaattccat atgatgacgc agcaccgct 29

<210> 7

<211> 32

<212> DNA

<213>primer (Primer)

<400> 7

ataagaatgc ggccgctcat tctaggctac ca 32

<210> 8

<211> 38

<212> DNA

<213>primer (Primer)

<400> 8

ggaattccat atggaaacta aatcccatca gaagcttc 38

<210> 9

<211> 45

<212> DNA

<213>primer (Primer)

<400> 9

ataagaatgc ggccgcttaa gacacaaaag acctcagctc ttcca 45

<210> 10

<211> 27

<212> DNA

<213>primer (Primer)

<400> 10

ctagctagca tggagagtaa gattcat 27

<210> 11

<211> 32

<212> DNA

<213>primer (Primer)

<400> 11

ataagaatgc ggccgctcag ctcaaccttc tg 32

<210> 12

<211> 33

<212> DNA

<213>primer (Primer)

<400> 12

cgggatccat ggcttctcaa gaatgccagc ttc 33

<210> 13

<211> 46

<212> DNA

<213>primer (Primer)

<400> 13

ataagaatgc ggccgcttag tttgtctttg tgcaatttcc ttgttg 46

<210> 14

<211> 38

<212> DNA

<213>primer (Primer)

<400> 14

ctagctagca tggcttctgg ttataatcat aagcctca 38

<210> 15

<211> 43

<212> DNA

<213>primer (Primer)

<400> 15

ataagaatgc ggccgcctag tacgtgcgct tccttaatag gac 43

<210> 16

<211> 41

<212> DNA

<213>primer (Primer)

<400> 16

gctgtttgta ttccatttcc tgctcaaagc cacattaagg c 41

<210> 17

<211> 33

<212> DNA

<213>primer (Primer)

<400> 17

aaatggaata caaacagcat gaggcttatg att 33

<210> 18

<211> 36

<212> DNA

<213>primer (Primer)

<400> 18

gaatcaggtg aagattcaac gcaagatatc actgtg 36

<210> 19

<211> 31

<212> DNA

<213>primer (Primer)

<400> 19

aatcttcacc tgattctgga aatccatcag g 31

<210> 20

<211> 45

<212> DNA

<213>primer (Primer)

<400> 20

caggtgaaga tccaattcaa gatatcactg tgcttattga atccg 45

<210> 21

<211> 32

<212> DNA

<213>primer (Primer)

<400> 21

aattggatct tcacctgatt ctggaaatcc at 32

<210> 22

<211> 39

<212> DNA

<213>primer (Primer)

<400> 22

gatccaacgc aagatcgcac tgtgcttatt gaatccgtc 39

<210> 23

<211> 31

<212> DNA

<213>primer (Primer)

<400> 23

cgatcttgcg ttggatcttc acctgattct g 31

<210> 24

<211> 44

<212> DNA

<213>primer (Primer)

<400> 24

gatatcactg tgcttctgga atccgtcaga atccatcttt tggc 44

<210> 25

<211> 30

<212> DNA

<213>primer (Primer)

<400> 25

cagaagcaca gtgatatctt gcgttggatc 30

<210> 26

<211> 31

<212> DNA

<213>primer (Primer)

<400> 26

ccagtgactt gcattatttc agatggtttc a 31

<210> 27

<211> 32

<212> DNA

<213>primer (Primer)

<400> 27

taatgcaagt cactggagaa ctagtactgt tg 32

<210> 28

<211> 33

<212> DNA

<213>primer (Primer)

<400> 28

ttttcagatg gtttcctttc cgcttttacc acc 33

<210> 29

<211> 33

<212> DNA

<213>primer (Primer)

<400> 29

aaggaaacca tctgaaaaaa tgcaagtcac tgg 33

<210> 30

<211> 33

<212> DNA

<213>primer (Primer)

<400> 30

ttcagatggt ttcatgtccg tttttaccac cag 33

<210> 31

<211> 33

<212> DNA

<213>primer (Primer)

<400> 31

acggacatga aaccatctga aaaaatgcaa gtc 33

<210> 32

<211> 45

<212> DNA

<213>primer (Primer)

<400> 32

cttggaattc ctattatgtt gatctacact atttctgctt gcagc 45

<210> 33

<211> 28

<212> DNA

<213>primer (Primer)

<400> 33

cataatagga attccaagtt cctcggca 28

<210> 34

<211> 36

<212> DNA

<213>primer (Primer)

<400> 34

ggaattccta ttgcaatgat ctacactatt tctgct 36

<210> 35

<211> 30

<212> DNA

<213>primer (Primer)

<400> 35

cattgcaata ggaattccaa gttcctcggc 30

<210> 36

<211> 39

<212> DNA

<213>primer (Primer)

<400> 36

attcctattg cattgtatta cactatttct gcttgcagc 39

<210> 37

<211> 30

<212> DNA

<213>primer (Primer)

<400> 37

atacaatgca ataggaattc caagttcctc 30

<210> 38

<211> 37

<212> DNA

<213>primer (Primer)

<400> 38

cctattgcat tgatctggac tatttctgct tgcagct 37

<210> 39

<211> 31

<212> DNA

<213>primer (Primer)

<400> 39

ccagatcaat gcaataggaa ttccaagttc c 31

<210> 40

<211> 33

<212> DNA

<213>primer (Primer)

<400> 40

ctattgcatt gatctacact ctttctgctt gca 33

<210> 41

<211> 33

<212> DNA

<213>primer (Primer)

<400> 41

gagtgtagat caatgcaata ggaattccaa gtt 33

<210> 42

<211> 29

<212> DNA

<213>primer (Primer)

<400> 42

actatttctg cttgcggctt catgggatt 29

<210> 43

<211> 33

<212> DNA

<213>primer (Primer)

<400> 43

cgcaagcaga aatagtgtag atcaatgcaa tag 33

<210> 44

<211> 30

<212> DNA

<213>primer (Primer)

<400> 44

tgcagcttca tgggatttag gaaattccgc 30

<210> 45

<211> 29

<212> DNA

<213>primer (Primer)

<400> 45

aaatcccatg aagctgcaag cagaaatag 29

<210> 46

<211> 51

<212> DNA

<213>primer (Primer)

<400> 46

ttatgggatc taccactgtt ttgccgaact acaaatcctg atgacatcat g 51

<210> 47

<211> 38

<212> DNA

<213>primer (Primer)

<400> 47

cagtggtaga tcccataatc ggatatcttt ctttctcg 38

<210> 48

<211> 35

<212> DNA

<213>primer (Primer)

<400> 48

tgggatctac caagcgattg ccgaactaca aatcc 35

<210> 49

<211> 38

<212> DNA

<213>primer (Primer)

<400> 49

atcgcttggt agatcccata atcggatatc tttctttc 38

<210> 50

<211> 34

<212> DNA

<213>primer (Primer)

<400> 50

gatctaccaa gcttttggcg aactacaaat cctg 34

<210> 51

<211> 33

<212> DNA

<213>primer (Primer)

<400> 51

ccaaaagctt ggtagatccc ataatcggat atc 33

<210> 52

<211> 35

<212> DNA

<213>primer (Primer)

<400> 52

cctgatgaca tcatgctgca ctacaccatg gaagc 35

<210> 53

<211> 32

<212> DNA

<213>primer (Primer)

<400> 53

cagcatgatg tcatcaggat ttgtagttcg gc 32

<210> 54

<211> 37

<212> DNA

<213>primer (Primer)

<400> 54

gacatcatgt tccactttac catggaagct gctgaag 37

<210> 55

<211> 35

<212> DNA

<213>primer (Primer)

<400> 55

aaagtggaac atgatgtcat caggatttgt agttc 35

<210> 56

<211> 29

<212> DNA

<213>primer (Primer)

<400> 56

gtcaattttg gcagcaccgc ggttttgac 29

<210> 57

<211> 36

<212> DNA

<213>primer (Primer)

<400> 57

ggtgctgcca aaattgacat aaacaattga gtttgg 36

<210> 58

<211> 33

<212> DNA

<213>primer (Primer)

<400> 58

tctcacacac agtggttggg gttcaatcat tga 33

<210> 59

<211> 33

<212> DNA

<213>primer (Primer)

<400> 59

ccccaaccac tgtgtgtgag aaatcctcca act 33

<210> 60

<211> 37

<212> DNA

<213>primer (Primer)

<400> 60

gctgtgttgg ccattcagcg cagaccagca aacaaac 37

<210> 61

<211> 27

<212> DNA

<213>primer (Primer)

<400> 61

gctgaatggc caacacagca tcggcac 27

<210> 62

<211> 42

<212> DNA

<213>primer (Primer)

<400> 62

tgttggccat tcttttggga ccagcaaaca aacagttact ac 42

<210> 63

<211> 30

<212> DNA

<213>primer (Primer)

<400> 63

ccaaaagaat ggccaacaca gcatcggcac 30

Claims (17)

1. isolated polypeptide, which is characterized in that the polypeptide is selected from:

(a) there is ammonia shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 The polypeptide of base acid sequence；

(b) in the space structure of the polypeptide of the amino acid sequence shown in SEQ ID NO:1, the 21st and the 121st amino acids are not Become, but is located near the 21st and the 121st amino acids position or occurs in substrate receptor binding pocket amino acid residue variation Polypeptide；

(c) on the basis of the amino acid sequence shown in SEQ ID NO:1, the 16th, 82,83,86,90,119,124,126,140, 141,142,143,145,149,153,198,199,200,210,212,302,379,397 or 398 generation amino acid variations Polypeptide；Preferably, P82S, T83I, I86R, I90L, F119I, A126V, A140M, L141M, S149G, L153F occurs, The polypeptide of Y212F or I302T variation；

(d) (a) or conservative variation's polypeptides (b) or (c).

2. polypeptide as described in claim 1, which is characterized in that the conservative variation's polypeptides are selected from:

(1) as (a) or (b) or (c) described in any polypeptide replacing, missing or adding by one or more amino acid residues And formed, and the polypeptide with polypeptide (a) function；

(2) amino acid sequence of the polypeptide of amino acid sequence with (a) or (b) or (c) has 80% or more the phase same sex, and has polypeptide (a) polypeptide of function；Or

(3) at (a) or (b) or (c), the N of the polypeptide or C-terminal add sequence label, or add signal peptide sequence in its N-terminal The polypeptide formed after column；

Preferably, the 21st is histidine in the conservative variation's polypeptides of SEQ ID NO:1, the 121st is asparatate.

3. polypeptide as claimed in claim 1 or 2, which is characterized in that the guarantor of the polypeptide of amino acid sequence shown in SEQ ID NO:1 Keeping property Variant polypeptides include: (b) or (c) in, the position of alanine near the 21st hyte propylhomoserin does not occur for the mutation.

4. polypeptide as claimed in claim 3, which is characterized in that (b) or (c) in, the polypeptide be the 16th, 82,83, 86、90、119、124、126、140、141、142、143、145、149、153、198、199、200、210、212、302、379、397 Or the polypeptide of 398 generation amino acid variations；Preferably, the polypeptide is that P82S, T83I, I86R, I90L, F119I occurs, The polypeptide of A126V, A140M, L141M, S149G, L153F, Y212F or I302T variation.

5. isolated polynucleotides, which is characterized in that it encodes any polypeptide of Claims 1 to 4.

6. a kind of carrier, which is characterized in that it contains polynucleotides described in claim 5.

7. a kind of genetically engineered host cell, which is characterized in that it contains carrier as claimed in claim 6 or its gene Polynucleotides described in claim 5 are integrated in group.

8. a kind of preparation method of any polypeptide of Claims 1 to 4, which is characterized in that this method includes: culture right It is required that host cell described in 7；Collect the culture containing any polypeptide of Claims 1 to 4.

9. the purposes of polypeptide described in claim 1, wherein the polypeptide be with SEQ ID NO:2, SEQ ID NO:3, The polypeptide or their conservative variation's polypeptides of amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5；For having A glycosyl (Glc) is shifted on the compound of parent nucleus (I) structure, forms the compound such as formula (II) structure:

Wherein, R is independently selected from H, OH, O- β-_DGlucosyl group, O- β-_DGlucosyl group-(2 → 1)-β-_DGlucosyl group.

10. purposes as claimed in claim 9, which is characterized in that the compound with parent nucleus (I) structure includes: sweet tea Alantol, Ent-kauran olefin(e) acid, steviol monoglycosides, steviol disaccharide glycosides；

The compound of described formula (II) structure includes: steviol -19-O- glucose ester, the Portugal Ent-kauran olefin(e) acid -19-O- Grape sugar ester, sweetness agent classification glycosides, stevioside.

11. purposes as described in claim 1, wherein the polypeptide has amino acid sequence shown in SEQ ID NO:1 Polypeptide or its conservative variation's polypeptides；For shifting a upper glycosyl (Glc) on the compound with parent nucleus (III) structure, Form the compound such as formula (IV) structure:

Wherein, R ' is independently selected from H, O- β-_DGlucosyl group.

12. purposes as claimed in claim 11, which is characterized in that the compound with parent nucleus (III) structure includes: Steviol -19-O- glucose ester, steviol；

The compound of described formula (IV) structure includes: sweetness agent classification glycosides, steviol monoglycosides.

13. the purposes as described in claim 9~12 is any, which is characterized in that the donor of the glycosyl is containing glycosyl Compound；Preferably, the donor includes but is not limited to: UDP-glucose, UDP- rhamnose, UDP- xylose；Or it is general It includes as UDP- sugar.

14. a kind of composition for glycosyl transfer, which is characterized in that it contain any polypeptide of Claims 1 to 4 or Contain host cell as claimed in claim 7, its culture or pyrolysis product.

15. the method that one kind shifts a upper glycosyl on the compound with parent nucleus (I) structure, which is characterized in that the side Method includes: the host cell that the polypeptide is expressed with polypeptide, its culture or pyrolysis product, or the composition containing the polypeptide will Glycosyl is transferred to the compound with parent nucleus (I) structure；

Wherein, R is independently selected from H, OH, O- β-_DGlucosyl group, O- β-_DGlucosyl group-(2 → 1)-β-_DGlucosyl group；

Wherein, which has amino acid shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 or SEQ ID NO:5 The polypeptide of sequence or their conservative variation's polypeptides.

16. the method that one kind shifts a upper glycosyl on the compound with parent nucleus (III) structure, which is characterized in that described Method includes: the host cell that the polypeptide is expressed with polypeptide, its culture or pyrolysis product, or the composition containing the polypeptide Glycosyl is transferred to the compound with parent nucleus (III) structure；

Wherein, R ' is independently selected from H, O- β-_DGlucosyl group；

Wherein, which has the polypeptide or their conservative variation's polypeptides of amino acid sequence shown in SEQ ID NO:1.

17. the method described in claim 16, which is characterized in that the donor of the glycosyl is the compound containing glycosyl； Preferably, the donor includes: UDP-glucose, UDP- rhamnose, UDP- xylose；Or it is summarised as UDP- sugar.